The field of the present invention includes molecular biology, in particular, with respect to the genetics and molecular mechanism of antibiotic resistance, especially macrolide resistance, in bacteria, and to methods for identifying inhibitors of macrolide resistance, in particular, that resistance to macrolides and/or streptogramins due to efflux systems.
Infections caused by Gram positive bacteria including but not limited to Streptococcus pneumoniae remain a devastating and worldwide health problem. The treatment of infections due to S. pneumoniae has become increasingly complicated due to the rapid emergence of resistance to penicillin. While macrolides (including, but not limited to, erythromycin, clarithromycin, azithromycin) are considered alternatives to penicillin for non-meningeal infections, resistance has rapidly emerged to these agents as well.
There are two known mechanisms of macrolide resistance in S. pneumoniae, target modification and macrolide efflux. The pneumococcal ermAM gene product, methylates highly conserved adenine residues in the peptidyl transferase center of newly synthesized 23S rRNA [6]. This methylation blocks the binding of macrolides, lincosamides and streptogramins B, thereby conferring the MLSB phenotype of antibiotic resistance [7]. The pneumococcal ermAM gene is associated with conjugative transposons that co-harbor other antimicrobial resistance determinants, e.g., tetM [8]. Until 1996 the only known form of macrolide resistance in pneumococci was due to a methylase (the ermAM gene product) which methylates highly conserved adenine residues in the peptidyl transferase center, domain V, of newly synthesized 23S rRNA. This methylation blocks the binding of macrolides, lincosamides and streptogramin B. The expression of this MLSB phenotype can be inducible or constitutive. There are at least eight classes of erm; ermA, B and C are found on the transposons found in several clinically important pathogens. The pneumococcal ermAM gene is associated with large conjugative and composite transposons, which coharbor other antimicrobial resistance determinants, e.g., tetM and cat. The ermAM gene is very similar to ermB [2, 24]; ermA and C have not been described in S. pneumoniae. Erythromycin MICs for pneumococci containing ermAM are typically=64 μg/ml. In certain other organisms, inactivation is an additional potential mechanism.
In 1996, a newly detected macrolide efflux mechanism, mefE, was identified in macrolide-resistant strains of S. pneumoniae lacking an ermAM determinant [9]. Pneumococcal strains containing mefE were reported to express resistance only to 14- and 15-membered macrolides (M phenotype). A 3.7 kb pneumococcal fragment containing mefE when cloned in E. coli was reported to encode a proton motive force-driven transporter sufficient to confer the M phenotype [10]. mefE and the related determinant mefA, originally described in S. pyogenes, are ˜90% identical and have been placed in a single class of macrolide efflux genes [11,12]. Both mefE and mefA are now found in S. pneumoniae [13,14] and are known to be transferable [15].
In Europe, the ermAM determinant is reported to account for recent increases in macrolide resistance of S. pneumoniae while mefA and mefE are found less often [16]. In contrast, most macrolide-resistant pneumococcal strains in North America harbor mefE [13,17-19]. In metropolitan Atlanta, between 1994 and 1999, macrolide resistance of invasive pneumococcal isolates increased from 16.4% to 31.5% [13]. By 1999, mefE was found in 26% of all invasive S. pneumoniae isolated in metropolitan Atlanta [13].
There is a need in the art for an understanding of the mechanisms of antibiotic resistance and the spread thereof, and for methods for identifying inhibitors of resistance mechanisms as well as for identifying compounds which evade the resistance mechanisms to allow for efficacious treatment of infectious diseases of bacterial origin.